Endogenous T-cell Research Articles

CD40 Ligand-Modified Chimeric Antigen Receptor T Cells Enhance Antitumor Function by Eliciting an Endogenous Antitumor Response

Chimeric antigen receptor (CAR) Tcells provide great efficacy in B cell malignancies. However, improved CAR Tcell therapies are still needed. Here, we engineered tumor-targeted CAR Tcells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR Tcells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR Tcells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing Tcells. These effects were absent in Cd40-/- mice and provide a rationale for the use of CD40L+ CAR Tcells in cancer treatment.

Abstract A171: A fully human tissue-based ex vivo cell migration analysis model to study T-cell infiltration and distribution in colorectal cancer liver metastases

Abstract T-cell exclusion by tumors is one of the major obstacles for cancer immunotherapies. In colorectal cancer liver metastasis (CRC-LM) patients, effector T-cells are mainly found in the invasive margin and not in the tumor epithelium. Such T-cell exclusion might hinder an effective antitumor immune response and could account for therapy failures with immune checkpoint inhibitors, which is observed in the majority of CRC-LM patients. As T-cell infiltration and distribution are difficult to detect in patients, little is known about detailed immunotherapy effects on tumor-infiltrating lymphocytes (TIL). Therefore, we established a fully human tissue-based ex vivo cell migration analysis model to monitor T-cell infiltration and positioning in the authentic tumor microenvironment of CRC-LM patients. In brief, we isolated T-cells from fresh resected CRC-LM patient tissue samples, labeled the cells with a fluorescent dye and returned the labeled T-cells to the tissues, which were cultured for a certain time period. Besides autologous T-cells, several tissue samples were treated with non-autologous labeled T-cells isolated from a healthy donor. After tissue processing, immunostaining and cell quantification, we observed that remigration of the labeled autologous and donor T-cells into the tissues was significantly associated with numbers of endogenous TIL. The labeled autologous and donor T-cells showed a similar distribution pattern to endogenous TIL with highest densities in the invasive margin of patient tissues. No relevant changes in tumor cell numbers or apoptosis protein concentrations could be observed by both treatments. Furthermore, quantification of the chemokines CXCL9, CXCL10 and CCL5 showed significant associations with labeled autologous and donor T-cell infiltration. Interestingly, treatment with a PD1 inhibitor did not support infiltration of exogenous or endogenous T-cells into the tumor epithelium. Our findings highlight that infiltrating T-cells in CRC-LM are always positioned into the invasive margin independently of the T-cell´s origin. Especially microenvironmental factors such as CXCL9, CXCL10 and CCL5 seem to be involved in this process, preventing T-cell contact with the tumor epithelium, which could also not be abrogated by immune checkpoint inhibition. The similar infiltration and distribution pattern of T-cells in the ex vivo and in vivo settings highlights the functionality and reliability of the human tissue-based cell migration analysis model, emphasizing its use for studying therapy effects on TIL in CRC-LM patient tissues. Citation Format: Anna Berthel, Meggy Suarez-Carmona, Jakob N. Kather, Rodrigo Rojas-Moraleda, Pornpimol Chaorentong, Nektarios A. Valous, Fee Klupp, Martin Schneider, Alexis Ulrich, Markus Buechler, Inka Zoernig, Dirk Jaeger, Niels Halama. A fully human tissue-based ex vivo cell migration analysis model to study T-cell infiltration and distribution in colorectal cancer liver metastases [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A171.

Abstract A188: T-cell priming with Deep IL-15 improves preclinical safety compared to systemic IL-15, and increases in vivo persistence and activity

Abstract Introduction: Interleukin-15 (IL-15), provides strong activation of both CD8+ T-cells and NK cells, without regulatory T-cells activation, making it an attractive immune modulator in cancer therapy. Systemic delivery of IL-15 to patients has revealed dose-limited toxicities resulting primarily in expansion of NK cells. Preclinical data suggest that IL-15 immunotoxicity is mediated by hyperproliferation and activation of NK cells (Guo Y, J Immunol 2015). In this study, we investigate safety and efficacy of T-cells loaded with Deep IL-15 (Deep IL-15 Primed T-cells), in a syngeneic mouse model. Deep IL-15 is a multimer of chemically crosslinked IL-15/IL-15 Rα/Fc heterodimers (IL15-Fc) that is designed for T-cell loading prior to adoptive cell transfer with the aim of improving the therapeutic window by autocrine signaling to the primed cells without causing the immunotoxicologic effects normally associated with IL-15. Deep IL-15 is loaded on the T-cells and, upon crosslinker cleavage, releases IL15-Fc to stimulate the primed cells. This novel T-cell-based therapeutic approach enables autocrine T-cell activation and expansion, and limits systemic exposure to IL15-Fc, thus reducing associated toxicities. Methods: Deep IL-15 was synthesized by incubation of IL15-Fc with a crosslinking reagent. PMEL CD8+ T (PMEL) cells were isolated from B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J mice. PMEL cells carry a transgenic T-cell receptor specific for gp100, a protein expressed by B16-F10 melanoma cells. PMEL cells were activated, expanded, and loaded with Deep IL-15 to generate Deep IL-15 Primed PMEL (Deep-15 PMEL) cells. Deep-15 PMEL cells were transferred into naïve or B16-F10 tumor-bearing mice (10 x 106; 15 ug Deep IL-15/106 cells), and the toxicity of Deep-15 PMEL was compared with PMEL (10 x 106) co-injected with soluble IL15-Fc at the maximum tolerated dose of 10 μg/mouse (PMEL + IL15-Fc). Readouts included IL15-Fc systemic exposure (ELISA), cytokine release (Luminex), and changes in endogenous T-cells (complete blood counts, CBC; flow cytometry). In addition, the biodistribution and the antitumor activity of Deep-15 PMEL was evaluated in B16-F10 tumor-bearing mice. Results: Deep-15 PMEL cells, carrying 15-fold more IL15-Fc than PMEL + IL15-Fc, resulted in >300-fold lower systemic exposure to IL15-Fc and 30-fold lower circulating IFN-γ. Deep-15 PMEL did not affect CBCs and did not expand endogenous CD8+ nor NK cells. Conversely, IL15-Fc induced significant changes in CBCs and promoted expansion of both transferred and endogenous CD8+ (6.7-fold) and NK (18.2-fold) cells. Similar results were observed in both naïve and tumor-bearing mice. Deep-15 PMEL improved persistence of transferred cells across multiple tissues (15-44-fold rel. to PMEL co-injected with IL15-Fc; day 16): blood, spleen, lymph nodes (tumor draining and non-draining) and tumor. The tumor presence affected the biodistribution of Deep-15 PMEL cells, resulting in lower (0.5-fold) accumulation of cells in the spleen, and increased (2.1-fold) cell numbers in the tumor-draining lymph node. Furthermore, Deep-15 PMEL showed significantly improved anti-tumor activity compared to PMEL in the B16-F10 model. Conclusions: The Deep Priming technology developed by Torque offers the advantage of loading Deep IL-15 prior to cell infusion in ACT, at concentrations unachievable with systemic injection of IL15-Fc, thus resulting in controlled, local delivery and cell-specific activation and proliferation. In vivo transfer of Deep-15 PMEL cells was well tolerated. Importantly, Deep-15 PMEL cells persisted longer across multiple organs compared to PMEL cells exposed to systemic IL15-Fc, while not inducing expansion of endogenous cells, including NK cells, which are reported to be the primary mediators of IL-15 toxicity. Torque is preparing to initiate clinical trials for Deep IL-15 Primed multitargeted human T-cells, TRQ15-01, in both hematologic and solid tumors. Citation Format: Elena Geretti, Philip Bardwell, Xiaoyan Liang, Santina Caruso, De-Kuan Chang, Jesse Lyons, Austin Boesch, Aaron Handler, Carlos Tassa, Sanela Bilic, Janice Lancita, Becker Hawes, Jonathan Fitzgerald, Thomas Andresen. T-cell priming with Deep IL-15 improves preclinical safety compared to systemic IL-15, and increases in vivo persistence and activity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A188.

Sintilimab: First Global Approval.

Sintilimab (Tyvyt®) is a fully human IgG4 monoclonal antibody that binds to programmed cell death receptor-1 (PD-1), thereby blocking the interaction of PD-1 with its ligands (PD-L1 and PL-L2) and consequently helping to restore the endogenous antitumour T-cell response. It has been co-developed by Innovent Biologics and Eli Lilly and Company, and was recently approved in China for the treatment of classical Hodgkin's lymphoma in patients who have relapsed or are refractory after ≥ 2 lines of systemic chemotherapy. Sintilimab is undergoing phase I, II and III development for use in various solid tumours, including non-small cell lung cancer and oesophageal cancer, in China. Phase I/II development of sintilimab for use in solid tumours is underway in the USA, with the US FDA accepting an Investigational New Drug application for sintilimab in January 2018. This article summarizes the milestones in the development of sintilimab leading to this first approval for the treatment of classical Hodgkin's lymphoma in patients who have relapsed or are refractory after ≥ 2 lines of systemic chemotherapy.

Abstract B205: Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients

Abstract Currently a TCR gene therapy trial to treat stage IV cutaneous and uveal melanoma patients is being conducted at the NKI. The TCR used for T-cell modification is specific for the HLA-A*0201 restricted MART-126-35 epitope, which is expressed on the majority of melanoma cells. Unique to this trial is the use of the combination of anti-CD3/CD28 beads for T-cell activation plus IL-7/ IL-15 for subsequent culture and expansion, instead of the more commonly used strategy that utilizes the combination of anti-CD3 and IL-2. The aim of this alternative production strategy is to generate a “less differentiated“ T-cell product that may have a better engraftment potential and antitumor activity. Thus far, a total of 12 patients have been treated, of whom 5 were uveal melanoma patients. 1 patient treated with 5x109 cells died 9 days after infusion, among others due to a severe cytokine release syndrome. The other 11 patients were treated in 3 different cell doses: 5x107, 25x107and10x107 transduced CD4 and CD8 T-cells. A prolonged engraftment and higher % of gene modified cells in the blood compartment were observed in patients treated with the highest number of cells infused. Gene modified T-cells could be found back in the circulation for up to two months post infusion, even when as few as 5x107 transduced T-cells were infused. In the highest cell dose, transduced T-cells could be detected up to 10 months following infusion. On-target toxicity against MART-1 expressing melanocytes in the skin (rash and vitiligo) was observed in all patients. Most severe skin toxicity, but also uveitis and inner ear toxicity, were observed in patients treated in the highest dose cohort of 2.5x107 transduced T-cells. These patients also developed a cytokine release syndrome requiring tocilizumab (anti-IL6R) treatment. Unexpected toxicity observed at this highest dose level consisted of development of pancytopenia around 6-8 weeks post T-cell infusion. In one of these patients, T-cell clonality of the infused cell product and of the peripheral blood compartment at the time of pancytopenia was analyzed in order to investigate the mechanism behind this toxicity. The highest clonality was found in the endogenous unmodified CD4 population. TCR diversity and evenness of the CD8 modified and unmodified populations were similar, suggesting that the pancytopenia was not associated with specific expansion of a TCR modified T-cell clone. However, this delayed pancytopenia could be related to 1) the conditioning chemotherapy regimen (fludarabine and cyclophosphamide), 2) cytokine release like IL-6, or 3) expansion of an autoreactive clone derived either from the infused T-cell product or newly generated from the endogenous T-cell pool. Of the 12 patients treated thus far, 8 patients had stable disease (SD) and two showed a partial response (PR) (5.2 and 9.5 month). The type of response or the duration of response was not correlated to the amount of cells infused or the peak and duration of engraftment of the infused T-cells. On the basis of these preliminary data, we conclude that TCR-modified T-cells produced by this method have a high engraftment potential and show in vivo activity at low infused cell doses, thereby suggesting the potential value of this protocol for TCR or CAR gene therapy for the treatment of cancer. Citation Format: Raquel Gomez-Eerland, Maartje Rohaan, Joost van den Berg, Maaike van Zon, Renate de Boer, Noor Bakker, Bastiaan Nuijen, Ton N.M. Schumacher, John B.A.G. Haanen. Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B205.

Abstract A109: Matrix metalloproteinase-2 stimulates Toll-like receptor-2 on melanoma cells to induce immunosuppressive inflammation in the tumor microenvironment

Abstract Extracellular proteinases, such as matrix metalloproteinases (MMPs), support tumor progression through modulation of the tumor microenvironment (TME). MMP-2, in particular, is overexpressed in several cancers and high MMP-2 levels are associated with advanced tumor stages, increased dissemination and poorer survival/prognosis. Our lab has previously demonstrated that upon antigenic stimulation, MMP-2-specific CD4+ T-cells, derived from patients with melanoma, secrete inflammatory TH2 cytokines. We subsequently showed that active MMP-2 drives the differentiation of TH2 responses by inhibiting IL-12 production and up-regulating OX40L expression on dendritic cells (DCs). We published our novel discovery identifying MMP-2 as a ligand for TLR-2 and showed that MMP-2 mediated TLR-2 stimulation lead to up-regulation OX40L on DCs (Cell Reports 2014). This is particularly interesting as TLR-2 stimulating adjuvants are being tested for immunotherapy. However, the full spectrum of how TLR-2 activation affects tumor cells or immune cells remains unclear.The main purpose of this study is to characterize the role of TLR-2-MMP-2 axis in shaping the TME through its influence on tumor cells and tumor infiltrating immune cells. Towards this end we performed RNA sequencing to identify genes induced in human DCs upon MMP-2 stimulation. One of these targets is an atypical member of the canonical NFκB family, IkappaBzeta (NFKBIZ or IκBζ). We show that MMP-2 secreted by melanoma cells upregulates IκBζ in DCs through TLR-2 and promotes secretion of Th2 and Th17 inducing cytokines. Furthermore, we screened several human melanoma cell lines for high and low MMP-2 and TLR-2 expression. CRISPR/Cas9 technology was used to stably knock out TLR-2, TLR-4 and MMP-2 in tumor cell lines. Early data indicates a role for tumor cell intrinsic MMP-2 in promoting secretion of protumorigenic cytokines and chemokines from tumor cells that support immune evasion and tumor growth, both constitutively and upon TLR-2 stimulation. Moreover, IκBζ was found to positively regulate MMP-2 dependent protumorigenic inflammation. In summary, we have identified a novel role for MMP-2 as a TLR-2 alarmin with particular emphasis on induction of atypical signaling modulator IκBζ and have uncovered a new role for MMP-2 in modulating tumor cell-induced inflammation. Taken together, our previous research and current data indicate that MMP-2 acts simultaneously as an endogenous T-cell differentiation "conditioner" and a tumor-associated antigen. Therefore, delving into MMP-2 signaling mechanisms in the TME holds a strong potential for discovering novel therapeutic options for treating melanoma. Citation Format: Mansi Saxena, Keerthi Caroline Sadanala, Luciana Rebiero Muniz-Bongers, Nina Bhardwaj. Matrix metalloproteinase-2 stimulates Toll-like receptor-2 on melanoma cells to induce immunosuppressive inflammation in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A109.

Abstract A043: Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro

Abstract Overall survival of pediatric B-precursor ALL patients reached 90% in recent years. However, the outcome for refractory or relapsed children remains very poor. Anti-CD19 chimeric antigen receptor T-cells (CD19-CAR) showed significant antileukemic activity in relapsed and refractory B-precursor ALL. Especially in children, isolation of a suitable T-cell amount for autologous CAR T-cell manufacturing can be challenging due to low blood volume, low T-cell counts and clinical condition. In this case, the adoptive transfer of CAR T-cells from an unmatched healthy third-party donor provides a promising strategy. In order to prevent life-threatening graft-versus-host disease, a knockout (KO) of the endogenous T-cell receptor (TCR) has to be performed. Here, we generated CD19-CARs with a CRISPR/Cas9 mediated TCR KO, which remain highly functional and show strongly reduced alloreactivity compared to conventional CAR T-cells introduced into third-party T-cells. T-cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and activated via anti-CD3/anti-CD28 stimulation. Retroviral transduction of a second generation anti-CD19 CAR (containing CD3zeta and 4-1BB stimulatory domains) was performed, followed by CRISPR/Cas9 mediated KO of the T-cell receptor beta chain via electroporation. After eleven days of expansion in the presence of IL-7 and IL-15, cells were purified for TCR KO-CD19-CAR T-cells via magnetic separation. Finally, the cell product was analyzed for cellular characteristics, functionality and alloreactivity by flow cytometry. A mean transduction rate of 37% for CD19-CARs and 40% for TCR KO-CD19-CARs was reached as well as a mean TCR KO rate of 78%. Both CD19-CARs as well as TCR KO-CD19-CARs showed suitable amounts of CD4- (45% vs. 33%) and CD8-T-cells (37% vs. 48%). The phenotype of CD19-CARs and TCR KO-CD19-CARs were comparable with mainly central memory (CM) (38% vs. 40%) and effector memory (EM) (57% vs. 51%) T-cells. The expansion of TCR KO-CD19-CARs was significantly reduced compared to conventional CD19-CARs (54-fold vs. 109-fold). This effect was not mediated by the loss of the TCR, but due to electroporation procedure. While CD19-CARs with or without TCR KO showed almost no background expression of the activation marker CD25 (2% vs 1%), contact with CD19-expressing targeT-cells resulted in a comparable upregulation of CD25 in both groups (95% vs. 94%). Co-culture with a CD19-expressing targeT-cell line led to an increased Interferon-γ secretion compared to unstimulated CARs, which was not significantly altered by the TCR KO (17% CD19-CAR vs. 14% TCR KO-CD19-CAR). CD19-dependent proliferative capacity of CAR T-cells was not influenced by loss of the TCR, as in both cases 97% of the T-cells proliferated after antigen recognition. Both CD19-CARs as well as TCR KO-CD19-CARs showed high, antigen-specific killing of 86% vs. 87% of the CD19-expressing targeT-cells at a 1:1 effector to target ratio. To evaluate the alloreactive potential of those T-cells, T-cells were co-cultured with irradiated PBMCs pooled from six different donors. 20% of TCR-expressing T-cells showed proliferation upon contact with non-HLA-matched PBMCs, whereas T-cells with a TCR KO showed almost no proliferation (<3%), demonstrating significantly reduced alloreactivity of TCR KO T-cells. CD19-CAR T-cells lacking the endogenous TCR show a balanced CD4 to CD8 ratio and high proportion of the favorable CM T-cell phenotype. TCR KO-CD19-CARs remain highly functional and show similar activation, cytotoxicity, proliferative capacity and cytokine secretion as conventional CD19-CARs upon antigen recognition. T-cells with TCR KO do not mediate an alloreactive response to non-HLA-matched PBMCs and therefore are promising candidates for the generation of CAR T-cells derived from nonmatched healthy third-party donors or for use of donor-derived CAR T-cells after haploidentical stem cell transplantation. Citation Format: Dana Stenger, Tanja Stief, Theresa Käuferle, Semjon Manuel Willier, Felicitas Rataj, Kilian Schober, Ramin Lotfi, Beate Wagner, Dirk H. Busch, Sebastian Kobold, Franziska Blaeschke, Tobias Feuchtinger. Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A043.

Abstract B139: Plant viral particle vaccine induces a potent antitumor response through induction of antigen-specific T-cells and overcoming an immunosuppressive tumor microenvironment

Abstract In order to develop an effective vaccination strategy for cancer, it is necessary to induce robust T-cell responses and to overcome the immunosuppressive tumor microenvironment (TME). To induce anticancer immunity, we focused on a plant viral particle (PVP) that contains-single strand RNA (ssRNA) as a flexible and economical platform to deliver cancer antigens. Initially using PVP loaded with SIINFEKL, we demonstrated the induction of a rapid CD8 T-cell response leading to a significant therapeutic effect in the B16 tumor model. The induction of high levels of CD8 T-cells was also achieved when clinically relevant cancer antigens were used with lysis of human cancer cells expressing these antigens. Further, PVP loaded with whole protein antigen induced CD8 and CD4 T-cell responses against the epitopes delivered through the whole antigen and these cleared established tumors. The mechanisms behind the high immunogenicity of the PVP vaccine revealed that this ssRNA containing vaccine activated the immune mechanisms closely resembling the natural antiviral defence. In a murine model, the immune induction was through the activation of TLR7, leading to release of type I IFN by plasmacytoid dendritic cells and activation of conventional DCs (cDCs) followed with the priming of Th1 immunity. These findings were further supported by experiments using human monocytes derived DCs (moDCs) stimulated in vitro with PVP. The stimulated moDCs released Th1-polarizing cytokines and chemokines, and upregulated CD40 and B7 family surface molecules. Further investigation of the TME demonstrated that cDCs, MHC-II high tumor associated macrophages (TAMs) and tumor-infiltrating neutrophils were recruited into the tumor bed 24 hours after vaccination. While MHC-II low TAMs and myeloid-derived suppressor cells were down-regulated in response to vaccination. This was followed by the increase of tumor-infiltrating lymphocytes (TILs) specific for endogenous tumor antigens. In conclusion, the PVP vaccine is an effective platform to induce T-cells specific to the delivered cancer antigens, and furthermore benefit from its ability to convert an immunosuppressive TME towards an immunostimulatory environment, facilitating the endogenous T-cell epitopes spread. Citation Format: Chuan Wang, Yidao Wang, Alex J. Allen, Jantipa Jobsri, Gareth J. Thomas, Christian H. Ottensmeier, Natalia Savelyeva. Plant viral particle vaccine induces a potent antitumor response through induction of antigen-specific T-cells and overcoming an immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B139.

Blinatumomab in the Treatment of Acute Lymphoblastic Leukemia: Russian Multicenter Clinical Trial

Background. Recent advances in the treatment of relapsed/refractory acute lymphoblastic leukemia (R/R ALL) are attributed to the implementation of immunotherapy methods which include blinatumomab, the bispecific engager of a patient's endogenous T-cells (Blincyto™, Amgen®) (BC). Aim. To assess BC efficacy and toxicity in the treatment of R/R ALL patients with persistence of minimal tumor clone before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Materials & Methods. The trial included 66 B-ALL patients with CD19+ aged 18 to 72 years, 23 (35 %) of them with measurable minimal residual disease (MRD+) and 43 (65 %) with R/R ALL. In 18 (27 %) patients BC was administered after prior allo-HSCT. Results. In the overall group 2-year overall survival (OS) and disease-free survival (DFS) in patients with response to BC treatment were 53 % and 38 % respectively. In the R/R ALL group complete remission (CR) was achieved in 29 (67 %) patients including 24 (83 %) patients with negative MRD. CR rate was higher in standard cytogenetic risk group (73 %) in comparison with high-risk group (59 %). In patients with more or less than 50 % blast cells in bone marrow CR rate was 85 % and 61 %, respectively. When BC was administered after prior allo-HSCT and without it CR rate was 80 % and 60 %, respectively. In R/R ALL patients with response to BC 2-year OS and DFS were 40 % and 26 %, respectively, in the MRD+ group of ALL patients they were 66 % and 51 %, respectively. Relapse rate was lower in the group with allo-HSCT than in the group without it, i.e. 21 % vs. 55 %. Adverse events of grade 3-4 were observed in 25 (38 %) patients. In 11 (16 %) patients BC therapy had to be discontinued, in 5 (7 %) patients it was terminated prior to the scheduled date. Conclusion. BC efficacy is higher in the MRD+ group and in R/R ALL patients with smaller tumor mass. BC treatment after allo-HSCT yields remissions in most patients and can be combined with immune-adoptive therapy.

An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.

: Effective immunotherapy of stromal-rich tumors requires simultaneous targeting of cancer cells and immunosuppressive elements of the microenvironment. Here, we modified the oncolytic group B adenovirus enadenotucirev to express a stroma-targeted bispecific T-cell engager (BiTE). This BiTE bound fibroblast activation protein on cancer-associated fibroblasts (CAF) and CD3ε on T cells, leading to potent T-cell activation and fibroblast death. Treatment of fresh clinical biopsies, including malignant ascites and solid prostate cancer tissue, with FAP-BiTE-encoding virus induced activation of tumor-infiltrating PD1+ T cells to kill CAFs. In ascites, this led to depletion of CAF-associated immunosuppressive factors, upregulation of proinflammatory cytokines, and increased gene expression of markers of antigen presentation, T-cell function, and trafficking. M2-like ascites macrophages exhibited a proinflammatory repolarization, indicating spectrum-wide alteration of the tumor microenvironment. With this approach, we have actively killed both cancer cells and tumor fibroblasts, reversing CAF-mediated immunosuppression and yielding a potent single-agent therapeutic that is ready for clinical assessment. SIGNIFICANCE: An engineered oncolytic adenovirus that encodes a bispecific antibody combines direct virolysis with endogenous T-cell activation to attack stromal fibroblasts, providing a multimodal treatment strategy within a single therapeutic agent.

Ex Vivo Assessment of Tnb-383B, a Bcma-Bispecific Antibody, Against Primary Tumor and Endogenous T Cells from Relapsing Multiple Myeloma Patients

INTRODUCTION: The human B-Cell maturation antigen (BCMA) is a surface marker that is highly expressed on plasma cells and has been recognized as a novel target in multiple myeloma (MM). TNB-383B is a fully human bispecific monoclonal IgG4 antibody. TNB-383B consists of 2 heavy and 1 light chain(s) paired through knob-in-hole technology. The first heavy chain and a kappa light chain form the paratope to recognize and bind human CD3. The second heavy chain is comprised of two identical VH domains in sequence and targets human BCMA with high affinity and avidity. Herein, we describe the ability of TNB-383B to mediate killing of patient-derived tumor cell lysis by endogenous T-cells was assessed ex vivo.METHODS: Bone marrow mononuclear cells (BMMCs) were isolated by density gradient centrifugation from 7 relapsed MM patients enrolled in an IRB-approved biospecimen collection protocol. Freshly isolated BMMC subsets were characterized by flow cytometry, specifically plasma cell (PC) / cytotoxic T cell (CTL) distribution, PC BCMA expression and PC viability were determined. BMMCs were then incubated for 24h (± 2h) with TNB-383B, or negative control, at concentrations ranging from 0.001-1μg. Following incubation, PC lysis, viability, BCMA expression, as well as CTL distribution and degranulation were assessed by flow cytometry. Tukey's sequential trend test was performed for each measured variable, utilizing ANOVA models and contrast statements, to detect linear dose response trends to TNB-383B or negative control treatments. Additionally, a parametric model (EMax) was used for each measured variable to estimate dose response curves, interpolating between tested doses. Two-way factorial ANOVA was utilized to compare the main effects of E:T ratio (or PC phenotype) and dose level and the interaction effect between E:T ratio and dose level on measured variables.RESULTS: Dose-dependent PC lysis was triggered by TNB-383B at concentrations as low as 0.001μg (p=0.0102) while no significant loss of PC was observed with negative control (Figure). This effect was coupled with significant CTL degranulation as expressed by increased CD107a mean fluorescence intensity (MFI) specific to TNB-383B treatment (p=0.0153 at 1μg). Although apoptotic rates (7-AAD+, Annexin V+) of the remaining PC tend to increase among TNB-383B treated compared with isotype control-treated cells, this trend was not significant. As opposed to CTL degranulation, CTL proliferation was not significantly triggered by TNB-383B but was significantly increased when BMMCs were exposed to negative control antibody (p=0.0057 at 0.001 μg). BMMC containing effector to target (E:T) ratio >10 contained lower viable (7-AAD-) PC and higher apoptotic PC counts compared with BMMC specimen with E:T ratio <10 (p<0.001). Using CD45 expression as a surrogate marker of PC maturation and BCMA expression, CD45+ PC displayed higher BCMA expression than CD45- PC (p=0.0189) and were more sensitive TNB-383B-induced killing (p<0.001). Noticeably, overall BCMA expression pre/post TNB-383B exposure remained unaltered.CONCLUSION: Taken together, our findings demonstrate TNB-383B triggers primary PC lysis and CTL degranulation in a dose-dependent fashion ex vivo. The ex vivo bispecific monoclonal antibody assay employed in this study allowed us to identify underlying biological drivers of PC killing efficacy by TNB-383B and may provide a valuable preclinical platform to screen bispecific antibodies and clinical platform to identify mechanism of primary or acquired resistance to the drug. Enrollment of patients with relapsed/refractory MM into a phase I clinical trial with TNB-383B is expected in early 2019. [Display omitted] DisclosuresFoureau:Teneobio Inc.: Research Funding. Pham:Teneobio Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Buelow:Teneobio Inc.: Employment. Voorhees:Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Other: served on an IRC; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Amgen Inc.: Speakers Bureau; TeneoBio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding.

Mosunetuzumab, a Full-Length Bispecific CD20/CD3 Antibody, Displays Clinical Activity in Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma (NHL): Interim Safety and Efficacy Results from a Phase 1 Study

Mosunetuzumab, a Full-Length Bispecific CD20/CD3 Antibody, Displays Clinical Activity in Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma (NHL): Interim Safety and Efficacy Results from a Phase 1 Study

Benchmarking the Foreign Antigen Space of Human Malignancies

Mutational load varies widely between malignancies and has been used as a proxy for the immunological foreignness of human cancers. However, without well-defined reference points it is difficult to determine which human tumors can be considered sufficiently foreign to the T-cell-based immune system. We established a neo-antigen prediction pipeline that processes single nucleotide variants, indels and gene fusion events and established its precision in identifying T-cell-recognized antigens. We used this pipeline to benchmark immunological foreignness of human cancers against pathogens for which T-cell control has been established. We demonstrate that up to 50% of tumors, spanning 25 sites of origin, are more foreign than these pathogen benchmarks. In addition, we demonstrate that the neo-antigen repertoire of treatment-naive tumors is not detectably influenced by immune editing. These data suggest that immunotherapeutic strategies that enhance activity of the endogenous T-cell compartment may be of value for a large fraction of human cancers.

Programmed cell death protein 1 activation preferentially inhibits CD28.CAR–T cells

Targeted adoptive immunotherapy with engineered T cells is a promising treatment for refractory hematologic malignancies. However, many patients achieving early complete remissions ultimately relapse. Immunosuppressive ligands are expressed on tumor and supportive cells in the tumor microenvironment (TME). When activated, T cells express associated “checkpoint” receptors. Binding of co-inhibitory ligands and receptors may directly contribute to T-cell functional exhaustion. It is not known whether all T cells engineered to express chimeric antigen receptors (CARs) are subject to checkpoint-mediated regulation. It is also unknown whether distinct CAR signaling moieties modulate T-cell responsiveness to these inhibitory pathways. We have, therefore, directly compared functional co-inhibition in engineered T cells identically targeted to the tumor-associated antigen CD123, but distinct in their mode of T-cell activation: via the endogenous T-cell receptor (ENG), or downstream of CD28 or 41BB-containing CARs. In all cases, we have observed antigen-independent T-cell activation associated with upregulation of the co-inhibitory receptors programmed cell death protein 1 (PD-1, CD279), Tim-3 and Lag-3. Notably, CD28.CAR T cells were uniquely susceptible to PD-1/PD-L1 mediated checkpoint inhibition. Together, our data indicate that PD-1/PD-L1 checkpoint blocking agents may be considered clinically when CD28.CAR T cells do not perform optimally in human trials.

IL35 Hinders Endogenous Antitumor T-cell Immunity and Responsiveness to Immunotherapy in Pancreatic Cancer.

Although successes in cancer immunotherapy have generated considerable excitement, this form of treatment has been largely ineffective in patients with pancreatic ductal adenocarcinoma (PDA). Mechanisms that contribute to the poor antitumor immune response in PDA are not well understood. Here, we demonstrated that cytokine IL35 is a major immunosuppressive driver in PDA and potentiates tumor growth via the suppression of endogenous antitumor T-cell responses. The growth of pancreatic tumors in mice deficient for IL35 was significantly reduced. An analysis of tumor-infiltrating immune cells revealed a role for IL35 in the expansion of regulatory T cells and the suppression of CD4+ effector T cells. We also detected a robust increase in both the infiltration and activation of cytotoxic CD8+ T cells, suggesting that targeting IL35 may be an effective strategy to convert PDA from an immunologically "cold" to "hot" tumor. Although PDA is typically resistant to anti-PD-1 immunotherapy, we demonstrated robust synergistic reduction in tumor growth when IL35 deficiency was combined with anti-PD-1 treatment. These findings provide new insight into the function of IL35 in the pathogenesis of pancreatic cancer and underscore the potential significance of IL35 as a therapeutic target for use in combination immunotherapy approaches in this deadly malignancy. Cancer Immunol Res; 6(9); 1014-24. ©2018 AACR.

The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

Targeting Merkel Cell Carcinoma by Engineered T Cells Specific to T-Antigens of Merkel Cell Polyomavirus.

Purpose: The causative agent of most cases of Merkel cell carcinoma (MCC) has been identified as the Merkel cell polyomavirus (MCV). MCV-encoded T antigens (Tag) are essential not only for virus-mediated tumorigenesis but also for maintaining MCC cell lines in vitro MCV Tags are thus an appealing target for viral oncoprotein-directed T-cell therapy for MCC. With this study, we aimed to isolate and characterize Tag-specific T-cell receptors (TCR) for potential use in gene therapy clinical trials.Experimental Design: T-cell responses against MCV Tag epitopes were investigated by immunizing transgenic mice that express a diverse human TCR repertoire restricted to HLA-A2. Human lymphocytes genetically engineered to express Tag-specific TCRs were tested for specific reactivity against MCC cell lines. The therapeutic potential of Tag-specific TCR gene therapy was tested in a syngeneic cancer model.Results: We identified naturally processed epitopes of MCV Tags and isolated Tag-specific TCRs. T cells expressing these TCRs were activated by HLA-A2-positive cells loaded with cognate peptide or cells that stably expressed MCV Tags. We showed cytotoxic potential of T cells engineered to express these TCRs in vitro and demonstrated regression of established tumors in a mouse model upon TCR gene therapy.Conclusions: Our findings demonstrate that MCC cells can be targeted by MCV Tag-specific TCRs. Although recent findings suggest that approximately half of MCC patients benefit from PD-1 pathway blockade, additional patients may benefit if their endogenous T-cell response can be augmented by infusion of transgenic MCV-specific T cells such as those described here. Clin Cancer Res; 24(15); 3644-55. ©2018 AACR.

Abstract 4963: Targeting glutamine metabolism as a means of enhancing antitumor T-cell responses

Abstract Tumors possess specialized metabolic programming that facilitates their rapid growth. We have developed a novel glutamine antagonist (JHU-083) that not only demonstrates single-agent efficacy in a variety of mouse tumor models but also synergizes with anti-PD-1, adoptive cellular therapy (ACT) and A2aR antagonism to overcome resistance to immunotherapy and promote durable cures. Interestingly, we have observed that JHU-083 markedly enhances endogenous antitumor T-cell responses even in the absence of additional immunotherapy. Flow cytometry analyses of tumor-infiltrating lymphocyte (TIL) reveal markedly increased infiltration of CD8+ T cells with increased proliferative index (Ki67) and the generation of robust memory phenotypes in JHU-083 treated mice. Interestingly, Gene Set Enrichment Analyses (GSEA) using RNA-sequencing data revealed that CD8+ TIL from vehicle treated mice showed significantly enhanced expression of apoptotic transcriptional programs compared with TIL from mice treated with JHU-083. Consistent with flow cytometry data, GSEA showed CD8+ TILs from mice treated with glutamine antagonism expressed transcriptional programs characteristic of long-lived memory cells. To this end, vaccination experiments in mice treated with JHU-083 demonstrate that many of the phenotypic changes observed in TIL can be generated outside the TME and are due to direct effects of glutamine blockade on effector T cells. Our studies show that these transcriptional changes are associated with profound remodeling of the histone epigenetic code and correlate with decreased intracellular levels of α-ketoglutarate in response to glutamine antagonism. Supplementation with dimethyl α-ketoglutarate, a cell-permeable form of α-ketoglutarate, partially reverses phenotypic and epigenetic changes in CD8 T cells undergoing activation in the setting of glutamine antagonism. These findings are consistent with inhibition of a family of enzymes known as α-ketoglutarate dependent dioxygenases, which are responsible for a broad range of demethylation reactions, including histone and DNA demethylation. Overall, our work demonstrates that glutamine antagonism can directly inhibit tumor growth and survival while also reprogramming maladaptive antitumor T-cell responses to enhance endogenous antitumor immunity. Such observations support the development of our novel glutamine antagonists as both monotherapy and in combination with immunotherapy. Citation Format: Robert Leone, Judson Englert, Im-Meng Sun, Min-Hee Oh, Jesse Alt, Im-Hong Sun, Ada J. Tam, Pavel Majer, Rana Rais, Barbara Slusher, Jonathan Powell. Targeting glutamine metabolism as a means of enhancing antitumor T-cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4963.

Abstract 1537: Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS

Abstract We designed the ARTEMISTM T-cell signaling platform to be a safer ACT therapy by relying on endogenous T-cell signaling factors. We show evidence that the ARTEMISTM receptor disentangles efficacy from CRS and holds the potential to be a clinically safer therapy by preventing ACT-triggered CRS. Despite the remarkable curative potential of anti-CD19-CAR T cells the therapy continues to trigger a potentially life threatening cytokine release syndrome (CRS) that is characterized by excessive concentrations of inflammatory cytokines. We reasoned that by redesigning the T-cell activating antigen receptor to require signaling through the endogenous CD3 complex the T-cell could better regulate its cellular responses and thus reduce the risk of CRS when used as an adoptive T-cell transfer (ACT) therapy. Through protein engineering, we developed the ARTEMIS™ signaling platform which when coupled with Eureka's human anti-CD19 antibody, ET190L1, and expressed on primary T-cells, results in a significant reduction of cytokine release during antigen specific T-cell activation. When CAR vs. ARTEMIS™ comparisons were tested in vitro, ET190L1-ARTEMISTM T-cells and ET190L1-CD28z-CAR T-cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour in vitro killing assays, ARTEMIS™ T-cells secreted demonstrably less IL-2, IFN-γ, GM-CSF, and TNFα as compared to CAR T-cells. ARTEMISTM T-cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion compared to corresponding CAR T-cells. The ability of the anti-IL6 antibody tocilizumab to mitigate toxicity in patients that develop severe CRS in the clinic suggests that limiting IL-6 expression could improve the safety of adoptive T-cell therapies. However, T-cells are not efficient expressors of IL-6 and the vast majority of elevated IL-6 is produced by monocytes in response to activated CAR T-cells. To assay the effects of T-cell cytotoxicity on upregulation of cytokine expression within monocytes as a model for CRS, we co-cultured T-cells with CD19-positive ALL tumor cells on one side of a transwell and monocytes on the other side. After tumor cell lysis, RNA expression levels were measured from both T-cells and monocytes. Using ET190L1-ARTEMIS™ T-cells to kill NALM-6 tumor cells resulted in dramatically reduced levels of IL-6 and IL-5 mRNA upregulation within monocytes compared to that observed with CAR transduced T-cells. When tested in vivo against CD19+ xenografts mouse models, intravenous administration of ET190L1-ARTEMISTM T-cells caused efficient tumor regression. In agreement with the in vitro data, mice treated with ARTEMISTM T-cells released significantly lower levels of cytokines in their blood at 24 hours post dosing than mice treated with CAR T-cells. Citation Format: Hong Liu, Lucas H. Horan, Stephan A. Grupp, David M. Barrett, Cheng Liu. Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1537.

Dissecting Epstein-Barr Virus-Specific T-Cell Responses After Allogeneic EBV-Specific T-Cell Transfer for Central Nervous System Posttransplant Lymphoproliferative Disease.

Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) with central nervous system (CNS) involvement is a severe complication after solid organ transplantation. Standard treatment with reduction of immunosuppression and anti-CD20 antibody application often fails leading to poor outcome. Here, we report the case of an 11-year-old boy with multilocular EBV-positive CNS PTLD 10 years after liver transplantation. Complete remission was achieved by repeated intravenous and intrathecal anti-CD20 antibody rituximab administration combined with intrathecal chemotherapy (methotrexate, cytarabine, prednisone) over a time period of 3 months. Due to the poor prognosis of CNS PTLD and lack of EBV-specific T-cells (EBV-CTLs) in patient’s blood, we decided to perform EBV-directed T-cell immunotherapy as a consolidating treatment. The patient received five infusions of allogeneic EBV-CTLs from a 5/10 HLA-matched unrelated third-party donor. No relevant acute toxicity was observed. EBV-CTLs became detectable after first injection and increased during the treatment course. Next-generation sequencing (NGS) TCR-profiling verified the persistence and expansion of donor-derived EBV-specific clones. After two transfers, epitope spreading to unrelated EBV antigens occurred suggesting onset of endogenous T-cell production, which was supported by detection of recipient-derived clones in NGS TCR-profiling. Continuous complete remission was confirmed 27 months after initial diagnosis.